Abstract
The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxin-binding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.
Highlights
Voltage-gated sodium channels (VGSCs) ␣ subunit subtypes (Nav1.1–1.9) have been cloned and characterized from mammals [1]
Mutations were principally designed using the following rules: 1) charged residues were mutated to be neutral, 2) unique residues in the spider VGSC [19], which is presumably resistant to HWTX-IV, were introduced into hNav1.7, and 3) if an uncharged residue is conserved in hNav1.7 and the spider VGSC, it was substituted by a smaller side chain residue, either Ala or Cys (Fig. 1)
We investigated the molecular determinants of VGSC voltage sensor trapping by the tarantula toxin HWTXIV
Summary
Molecular Biology—All of the hNav1.7 mutations were constructed using the QuikChange II XL site-directed mutagenesis kit according to the manufacturer’s instruction. Whole cell patch clamp recordings were carried out at room temperature (ϳ21 °C) using an EPC-10 amplifier (HEKA, Lambrecht, Germany). The standard bathing solution was 140 mM NaCl, 3 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES, pH 7.3. Linear leak subtraction, based on resistance estimates from four to five hyperpolarizing pulses applied before the depolarizing test potential, was used for all voltage clamp recordings. Dose-response curves to determine IC50 values were fitted using the Hill equation: y ϭ 1/(1 ϩ exp((logIC50 Ϫ X)nH), where X is the toxin dose, nH is the Hill coefficient, and IC50 is the half-maximal inhibitory concentration. The nH was set to 1 because our mutagenesis data have shown that the toxin had a single high affinity binding site in sodium channels. For HWTX-IV action on fast inactivation of WT and mutant Nav1.7, the nH was set to 1 because only sodium channel DIV is involved in channel inactivation gating
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